Title: Supplemental details for materials and methods
(ms:1266526906468429)
Ophthaldialdehyde assay
The ophthaldialdehyde (OPA) reagent was prepared by adding 40 mg of
phthaldialdehyde dissolved in 1 ml of methanol (BDH Laboratory supplies, Leicester,
UK) to a solution of 100 mg of mercaptoethanesulfonate dissolved in 50 ml of 0.1M
pH 9.2 sodium tetraborate. A 20µl protein sample was added to a Costar™ dark flat
bottomed 96 well plate (Sigma, Dorset UK) and 300 µl of OPA reagent was added to
each sample. The samples were incubated for 3 min at ambient room temperature
before being sampled using an Omega Fluostar plate reader (BMG Labtech,
Aylesbury UK) at excitation wavelength 355 nm and emission wavelength 460 nm[9].
Protein standard curves
A standard curve of protein concentrations 1, 5,10,20,50,100 µg /ml of protein was
prepared in RO H2O. A blank control of RO H2O was also included in standard
curves when used to determine the sensitivity of the protein assays and values of
unknown samples.
Validation of protein extraction methods
Medical grade stainless steel discs that had been prepared as described previously
(Section) were inoculated with a 10 µl solution of 10 mg/ ml BSA for a final
concentration 100 µg / ml solution on the disc. Discs were then dried for 16h
overnight at ambient room temperature. Discs were subjected to one of the
following treatments; sonication for 30 mins in Decon 90 (Decon laboratories East
Sussex UK) detergent, sonication for 30 mins in 1% SDS, and boiling for 10 mins in
0.1M NaOH. Each disc was inserted into a sterile 10 ml Bijoux (place name) and
immersed in 1 ml of the appropriate detergent; turbines were inserted into a 25 ml
Universal tube and immersed in 2 ml of the appropriate detergent. For sonication,
each tube was inserted into a Fisherbrand® 11021 sonic bath (Fisher Scientific
Leicestershire UK) and subjected to sonication at 35 kHz for the appropriate time.
For boiling, the tubes were inserted into a water bath (Grant Instruments) set at
100 °C for the appropriate time. A sample of the eluent was taken for the OPA
assay and sampled as previously described . Discs were inoculated with RO H2O and
dried for 16h as negative controls. A sample was also taken for SDS –
polyacrylamide gel electrophoresis (PAGE).
Results
Protein assay standard curves
An increase in BSA protein concentration results in a corresponding linear increase
in fluorescent units (figure 1) for the OPA assay with the limit of detection being 5
Mean Fluorescent Units
µg /ml.
125000
100000
75000
50000
25000
0
0
25
50
75
100
125
Protein concentration (g / ml)
Figure 1 OPA Bovine serum albumin standard curves diluted in1 % SDS. The BSA standard.
The data shown is the average of 3 readings from 3 experiments ± the SEM. The r2 value is
calculated as 0.9834.
Validation of protein extraction methods
The method that recovered the most protein was sonication in 1% SDS (Table 1).
Boiling in 0.1M NaOH recovered the least protein. BSA extraction by sonication in
1% (v/v) was visualised by SDS-PAGE (Figure 2).
Table 1 Efficacy of protein extraction techniques and detergents. The data shown is the median
of three experiments.
Detergent
Extraction Process
Median protein
Range of protein
recovery (µg / ml)
recovery (µg / ml)
1 % (v/v) SDS
Sonication
80
63.7 – 94.9
1 % (v/v) Decon90
Sonication
57
22.8 – 77.8
0.1M NaOH
Boiling
53
50 – 66.5
Figure 2 Recovery of BSA by sonication and boiling technique. Both boiling (lanes 3 ,4 ,5) and
sonication (lanes 7, 8, 9) recover BSA from stainless steel discs. No protein is visible in the negative
controls (lane 2 and lane 6).